Alan W. Decho

The role of microbes in accretion, lamination and early lithification of modern marine stromatolites.

For three billion years, before the Cambrian diversification of life, laminated carbonate build-ups called stromatolites were widespread in shallow marine seas. These ancient structures are generally thought to be microbial in origin and potentially preserve evidence of the Earths earliest biosphere. Despite their evolutionary significance, little is known about stromatolite formation, especially the relative roles of microbial and environmental factors in stromatolite accretion. Here we show that growth of modern marine stromatolites represents a dynamic balance between sedimentation and intermittent lithification of cyanobacterial mats. Periods of rapid sediment accretion, during which stromatolite surfaces are dominated by pioneer communities of gliding filamentous cyanobacteria, alternate with hiatal intervals. These discontinuities in sedimentation are characterized by development of surface films of exopolymer and subsequent heterotrophic bacterial decomposition, forming thin crusts of microcrystalline carbonate. During prolonged hiatal periods, climax communities develop, which include endolithic coccoid cyanobacteria. These coccoids modify the sediment, forming thicker lithified laminae. Preservation of lithified layers at depth creates millimetre-scale lamination. This simple model of modern marine stromatolite growth may be applicable to ancient stromatolites.

Transfer of gold nanoparticles from the water column to the estuarine food web.

Within the next five years the manufacture of large quantities of nanomaterials may lead to unintended contamination of terrestrial and aquatic ecosystems. The unique physical, chemical and electronic properties of nanomaterials allow new modes of interaction with environmental systems that can have unexpected impacts. Here, we show that gold nanorods can readily pass from the water column to the marine food web in three laboratory-constructed estuarine mesocosms containing sea water, sediment, sea grass, microbes, biofilms, snails, clams, shrimp and fish. A single dose of gold nanorods (65 nm length x 15 nm diameter) was added to each mesocosm and their distribution in the aqueous and sediment phases monitored over 12 days. Nanorods partitioned between biofilms, sediments, plants, animals and sea water with a recovery of 84.4%. Clams and biofilms accumulated the most nanoparticles on a per mass basis, suggesting that gold nanorods can readily pass from the water column to the marine food web.

Ecotoxicity test methods for engineered nanomaterials: Practical experiences and recommendations from the bench

Ecotoxicology research is using many methods for engineered nanomaterials (ENMs), but the collective experience from researchers has not been documented. This paper reports the practical issues for working with ENMs and suggests nano-specific modifications to protocols. The review considers generic practical issues, as well as specific issues for aquatic tests, marine grazers, soil organisms, and bioaccumulation studies. Current procedures for cleaning glassware are adequate, but electrodes are problematic. The maintenance of exposure concentration is challenging, but can be achieved with some ENMs. The need to characterize the media during experiments is identified, but rapid analytical methods are not available to do this. The use of sonication and natural/synthetic dispersants are discussed. Nano-specific biological endpoints may be developed for a tiered monitoring scheme to diagnose ENM exposure or effect. A case study of the algal growth test highlights many small deviations in current regulatory test protocols that are allowed (shaking, lighting, mixing methods), but these should be standardized for ENMs. Invertebrate (Daphnia) tests should account for mechanical toxicity of ENMs. Fish tests should consider semistatic exposure to minimize wastewater and animal husbandry. The inclusion of a benthic test is recommended for the base set of ecotoxicity tests with ENMs. The sensitivity of soil tests needs to be increased for ENMs and shortened for logistics reasons; improvements include using Caenorhabditis elegans, aquatic media, and metabolism endpoints in the plant growth tests. The existing bioaccumulation tests are conceptually flawed and require considerable modification, or a new test, to work for ENMs. Overall, most methodologies need some amendments, and recommendations are made to assist researchers.

A laboratory investigation of cyanobacterial extracellular polymeric secretions (EPS) in influencing CaCO3 polymorphism

Bahamian stromatolites are well-laminated structures, consisting of lithified layers alternating between unlithified layers containing fine-grained carbonate ooids. The lithified layers consist of abundant aragonite needles embedded within a matrix of extracellular polymeric secretions (EPS) by cyanobacteria, Schizothrix sp. Laboratory investigations were conducted using EPS extracted from natural stromatolites and laboratory isolates of Schizothrix sp., to chemically characterize EPS, and determine in vitro how EPS may influence CaCO3 polymorphism. EPS mainly consisted of acidic polysaccharides and proteins. Biochemical analyses indicated that contents of uronic acids and carbohydrates in EPS from lithified layers decreased when compared with unlithified layer EPS, while the protein content remained relatively constant. CaCO3 nucleation experiments demonstrated that EPS from the lithified layer, induced aragonite crystal formation in vitro, as confirmed by scanning electron microscopy and Fourier transform infrared (FT-IR) spectroscopy. In contrast, EPS from the unlithified layer or laboratory-cultured Schizothrix sp. induced calcite crystal formation. These laboratory results suggest the possibility that the biochemical composition, specifically small proteins, of EPS influences the resulting mineralogy of CaCO3. r 2002 Elsevier Science B.V. All rights reserved.

Quorum sensing in natural environments: emerging views from microbial mats.

Much laboratory-based information exists on quorum sensing, a type of bacterial cell-to-cell communication that depends upon exchanges of molecular signals between neighboring cells. However, little is known about how this and other microbial sensing systems operate in nature. Geochemical and biological modifications of signals probably occur in extracellular environments, and these could disrupt intended communication if signals are no longer recognized. However, as we discuss here, signal alterations might result in other outcomes: if a modified signal is able to interact with a different receptor then further environmental information can be gained by the receiving cells. We also postulate that quorum sensing occurs within cell clusters, where signal dispersion might be significantly influenced by extracellular polymers. As a model system to discuss these points we use microbial mats - highly-structured biofilm communities living under sharply-defined, fluctuating geochemical gradients.

Antimicrobial metallopolymers and their bioconjugates with conventional antibiotics against multidrug-resistant bacteria.

Bacteria are now becoming more resistant to most conventional antibiotics. Methicillin-resistant Staphylococcus aureus (MRSA), a complex of multidrug-resistant Gram-positive bacterial strains, has proven especially problematic in both hospital and community settings by deactivating conventional β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems, through various mechanisms, resulting in increased mortality rates and hospitalization costs. Here we introduce a class of charged metallopolymers that exhibit synergistic effects against MRSA by efficiently inhibiting activity of β-lactamase and effectively lysing bacterial cells. Various conventional β-lactam antibiotics, including penicillin-G, amoxicillin, ampicillin, and cefazolin, are protected from β-lactamase hydrolysis via the formation of unique ion-pairs between their carboxylate anions and cationic cobaltocenium moieties. These discoveries could provide a new pathway for designing macromolecular scaffolds to regenerate vitality of conventional antibiotics to kill multidrug-resistant bacteria and superbugs.

Characteristics and turnover of exopolymeric substances in a hypersaline microbial mat

The properties and microbial turnover of exopolymeric substances (EPS) were measured in a hypersaline nonlithifying microbial mat (Eleuthera, Bahamas) to investigate their potential role in calcium carbonate (CaCO(3)) precipitation. Depth profiles of EPS abundance and enzyme activities indicated that c. 80% of the EPS were turned over in the upper 15-20 mm. Oxic and anoxic mat homogenates amended with low-molecular-weight (LMW) organic carbon, sugar monomers, and different types of EPS revealed rapid consumption of all substrates. When comparing the consumption of EPS with that of other substrates, only marginally longer lag times and lower rates were observed. EPS (5-8%) were readily consumed during the conversion of labile to refractory EPS. This coincided with a decrease in glucosidase activity and a decrease in the number of acidic functional groups on the EPS. Approximately half of the calcium bound to the EPS remained after 10 dialyses steps. This tightly bound calcium was readily available to precipitate as CaCO(3). We present a conceptual model in which LMW organic carbon complexed with the tightly bound calcium is released upon enzyme activity. This increases alkalinity and creates binding sites for carbonate and allows CaCO(3) to precipitate. Therefore, this model explains interactions between EPS and CaCO(3) precipitation, and underscores the critical role of aerobic and anaerobic microorganisms in early diagenesis and lithification processes.

Autoinducers extracted from microbial mats reveal a surprising diversity of N-acylhomoserine lactones (AHLs) and abundance changes that may relate to diel pH.

Microbial mats are highly structured and diverse communities, and one of the earliest-known life assemblages. Mat bacteria interact within an environment marked by strong geochemical gradients and fluctuations. We examined natural mat systems for the presence of autoinducers involved in quorum sensing, a form of cell-cell communication. Our results revealed that a diverse array of N-acylhomoserine lactones (AHLs) including C(4)- to C(14)-AHLs, were identified from mat extracts using mass spectrometry (MS), with further confirmation by MS/MS-collision-induced dissociation (CID), and additions of external standards. Microelectrode measurements showed that mats exhibited diel pH fluctuations, ranging from alkaline (pH 9.4) during daytime (net photosynthesis) to acidic (pH 6.8) during darkness (net respiration/fermentation). Under laboratory conditions, AHLs having shorter acyl-chains were degraded within the time frame that daily alkaline pH (> 8.2) conditions exist in mats. Intensive sampling of mats after full day- or night-time incubations revealed that accumulations of extractable shorter-chain AHLs (e.g. C(8)- and C(10)-AHLs) were significantly (P < 0.001) diminished during daytime. Our study offers evidence that stabilities of AHLs under natural conditions may be influenced by the proximal extracellular environment. We further propose that the ancient periodicity of photosynthesis/respiration in mats may potentially drive a mechanism for diel differences in activities of certain autoinducers, and hence bacterial activities mediated through quorum sensing.

Exopolymer Microdomains as a Structuring Agent for Heterogeneity Within Microbial Biofilms

It is now well-recognized that the majority, and often most active fractions, of microbial cells in many natural systems occur as surface-associated biofilms. In sedimentary environments, biofilm formation represents an important functional adaptation for microbial life. At the level of an individual sediment particle, the biofilm community represents a cacophony of cellular and extracellular processes enclosed within an amorphous biofilm. Recent studies using new analytical approaches now suggest that the seemingly amorphous biofilm instead may be a highly structured system, one in which microbial cells actively manipulate their extracellular polymers and overall microenvironment to accomplish specific tasks. At microspatial scales (nanometers to micrometers), biofilm polymers are important in sequestering of nutrients, localization of extracellular enzymes, and providing a protective and stabilizing microenvironment for cells. Examination of the three-dimensional nature of microbial biofilm communities and activities through the use of nuclear magnetic resonance (NMR) spectroscopy, confocal laser microscopy (CLM), atomic-force microscopy (AFM) and other techniques are beginning to provide quantitative evidence for microscale partitioning within biofilms. In light of these new data, the biofilm is explored here as an important structural matrix to partition microbial extracellular activities and effectively promote heterogeneity over very small (i.e., molecular) spatial scales. Structuring and partitioning may occur through the formation of “exopolymer-mediated microdomains.” These are regions of a biofilm matrix where specific types of exopolymers are concentrated and impart unique physical/chemical properties to the biofilm. Accumulating evidence, derived from isotope sorption studies, electron microscopy, and CLM supports this idea. The presence of exopolymer microdomains may provide microorganisms with a structuring mechanism to spatially segregate extracellular activities over small spatial scales.

Determining the specific microbial populations and their spatial distribution within the stromatolite ecosystem of Shark Bay

The stromatolites at Shark Bay, Western Australia, are analogues of some of the oldest evidence of life on Earth. The aim of this study was to identify and spatially characterize the specific microbial communities associated with Shark Bay intertidal columnar stromatolites. Conventional culturing methods and construction of 16S rDNA clone libraries from community genomic DNA with both universal and specific PCR primers were employed. The estimated coverage, richness and diversity of stromatolite microbial populations were compared with earlier studies on these ecosystems. The estimated coverage for all clone libraries indicated that population coverage was comprehensive. Phylogenetic analyses of stromatolite and surrounding seawater sequences were performed in ARB with the Greengenes database of full-length non-chimaeric 16S rRNA genes. The communities identified exhibited extensive diversity. The most abundant sequences from the stromatolites were α- and γ-proteobacteria (58%), whereas the cyanobacterial community was characterized by sequences related to the genera Euhalothece, Gloeocapsa, Gloeothece, Chroococcidiopsis, Dermocarpella, Acaryochloris, Geitlerinema and Schizothrix. All clones from the archaeal-specific clone libraries were related to the halophilic archaea; however, no archaeal sequence was identified from the surrounding seawater. Fluorescence in situ hybridization also revealed stromatolite surfaces to be dominated by unicellular cyanobacteria, in contrast to the sub-surface archaea and sulphate-reducing bacteria. This study is the first to compare the microbial composition of morphologically similar stromatolites over time and examine the spatial distribution of specific microorganismic groups in these intertidal structures and the surrounding seawater at Shark Bay. The results provide a platform for identifying the key microbial physiology groups and their potential roles in modern stromatolite morphogenesis and ecology.